Project Summary/Abstract Vaccines against infectious diseases have been hailed as the greatest achievement in public health over the last century. This competing renewal will investigate a novel adjuvant that elicits cellular immune responses needed for better vaccines. The lack of an appropriate adjuvant is one major barrier to developing a safe and effective vaccine against infections with fungal pathogens, which represents an unmet need in medicine and public health. Despite the rising rates of severe fungal infections, no vaccines against fungi are commercially available. While current vaccines against infectious diseases preferentially induce production of antibodies, their contribution to host defense against fungi is limited. Cellular immunity is essential for the resolution of fungal infections. Vaccine-induced resistance against fungi in experimental models requires Th17 and Th1 cells that produce IL-17 and IFN-? and mediate protection in part by recruiting and activating phagocytes to augment killing of fungi. In the last funding cycle, we discovered that fungal recognition by the C-type lectin receptor (CLR) Dectin-2 is required for the differentiation of protective Th17 and Th1 cells against dimorphic fungi. Through biochemical purification and mass spectrophotometry analysis, we identified a novel bona-fide fungal ligand for Dectin-2, the glycoprotein Blastomyces Eng3 (Bl-Eng3). In this application, we propose to characterize and functionally test Bl-Eng3 as an adjuvant for vaccination against fungi. We hypothesize that Bl-Eng3 activates dendritic cells, drives differentiation of antigen (Ag)-specific Th17 and Th1 cells, and thereby promotes protective cellular immunity against fungal infection. We also posit that the protein backbone and glycan modifications account for ligand activity and that Bl-Eng3 will be an effective adjuvant alone or together with other CLR ligands for vaccines against fungi. We provide strong preliminary data to support our hypotheses. Using Dectin-2 reporter cells, we have established an in vitro screening system to dissect the contributions of the protein backbone and its glycosylation to Dectin-2 ligand activity and an in vivo adoptive transfer system to delineate the effects of the ligand adjuvant on the differentiation of nave antigen-specific CD4+ T-cells into protective Th17 and Th1 cells. Our approach offers a powerful complimentary strategy that will identify the ligand moiety of the glycoprotein in aim 1, assess adjuvancy for the maturation and priming of Ag-presenting cells (APCs) and Ag-specific T cells in aim 2, and determine receptor collaboration with other CLRs and protective efficacy in experimental models of pulmonary and systemic fungal infections in aim 3. Our work will provide new insight needed to promote the maturation of APCs and differentiation of protective Th17 and Th1 cells. This knowledge will provide the basis for developing and designing new vaccine strategies against fungi, and other pathogenic microbes that require cellular immunity for host defense.